The present invention relates to a composition and method comprising Mycobacterium phlei (M. phlei)-DNA (M-DNA), M-DNA preserved and complexed on M. phlei cell wall (MCC) and a chemotherapeutic agent, wherein the M-DNA and the MCC are effective for treating cancer and for potentiating the antineoplastic effect of the chemotherapeutic agent on the cancer.
Cancer is an aberrant net accumulation of atypical cells, which results from an excess of proliferation, an insufficiency of cell death, or a combination of the two.
Proliferation is the culmination of a cell""s progression through the cell cycle and is characterized by replication of total cellular DNA and the division of one cell into two cells. For cell division, mammalian cells pass through an organized series of controlled events, referred to as the cell cycle. The initiation of an event during cell cycle progression is dependent on the successful completion of an earlier event. The cell cycle can be divided into 5 major phases. These are Go, G1, S, G2 and M. During the Go phase, cells are quiescent. Most cells in the body, at any one time, are in this stage. During the G1 phase, cells, responding to signals to divide, produce the RNA and the proteins necessary for DNA synthesis. During the S-phase (SE, early S-phase; SM, middle S-phase; and SL, late S-phase) the cells replicate their DNA. At the end of the S phase, each cell contains twice its original DNA content but is still bound by one external cell membrane. During the G2 phase, proteins are elaborated in preparation for cell division. During the mitotic (M) phase, the cell divides into two daughter cells.
Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes or abrogation of DNA damage checkpoints, and may modulate the cellular response to treatment with chemotherapeutic agents (Hochhauser D. Anti-cancer Chemotherapeutic Agents 8:903, 1997).
Cell death is effected by immune-mediators that initiate cytolytic processes and that promote apoptosis, and from apoptosis inducers that directly initiate pathways leading to cell death. Apoptosis is an active cellular death process characterized by distinctive morphological changes that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al. Int. Rev. Cytol. 68:251, 1980). A molecular hallmark of apoptosis is degradation of the cell""s nuclear DNA into oligonucleosomal-length fragments as the result of activation of endogenous endonucleases (Wyllie A. Nature 284:555, 1981).
Caspases have been implicated as key enzymes in the execution phase of apoptosis. The caspase family consists of at least fourteen related cysteine aspartane proteases. All the caspases contain a conserved QACXG (where X is R, Q or G) (SEQ ID NO: 1) pentapeptide active-site motif (Cohen G. Biochim. Biophys. Acta 1366:139, 1997). A number of caspases are synthesized as inactive proenzymes, which are activated following cleavage at specific aspartate cleavage sites (Cohen G. Biochim. Biophys. Acta 1366:139, 1997) or as inactive enzymes that require association with regulatory molecules for activation (Stennicke et al. J. Biol. Chem. 274:8359, 1999). Activation of the initiator procaspase activates downstream effector caspases triggering the cell death cascade (Pan et al. J. Biol. Chem. 273:5841, 1998; Earnshaw W. Nature 397:387, 1999).
Most currently used chemotherapeutic agents are nonspecifically cytotoxic. Many of these chemotherapeutic agents have toxic side effects, are debilitating and often compromise the quality of life of the patient. Moreover, alterations in the transport and metabolism of chemotherapeutic agents by the cancer cells result in the development of resistance to the chemotherapeutic agents by the cancer cells.
Therefore, there is a continuing need for novel compositions and methods that induce cell cycle arrest in cancer cells, that inhibit proliferation of cancer cells, that induce apoptosis in cancer cells and that potentiate the antineoplastic effect of chemotherapeutic agents on cancer cells. Moreover, such compositions should be simple and relatively inexpensive to prepare, their activity should remain therapeutically stable over time and they should be effective at dose regimens that are associated with minimal toxicity even upon repeated administration.
The present invention satisfies these needs by providing a composition and method comprising Mycobacterium phlei (M. phlei)-DNA (M-DNA), M-DNA preserved and complexed on M. phlei cell wall (MCC). a chemotherapeutic agent and a pharmaceutically acceptable carrier, wherein the M-DNA and the MCC induce cell cycle arrest in cancer cells, inhibit proliferation of cancer cells, induce apoptosis in cancer cells and potentiate the antineoplastic effect of the chemotherapeutic agent on cancer cells. Moreover, M-DNA and MCC are simple and relatively inexpensive to prepare, their activity is reproducible among preparations, remains therapeutically stable over time, and is effective at dose regimens that are associated with minimal toxicity even upon repeated administration.
To prepare MCC, M. phlei are grown in liquid medium and harvested. The M. phlei are disrupted, and the solid components of the disrupted M. phlei are collected by centrifugal sedimentation. The solid components are deproteinized, delipidated, and washed. All reagents are selected to enhance conservation of DNA during MCC preparation. M-DNA is prepared from MCC or directly from M. phlei. Again, all reagents are selected to enhance conservation of DNA during M-DNA preparation.
A composition comprising M-DNA, MCC, M-DNA+ chemotherapeutic agent or MCC+ chemotherapeutic agent is administered to an animal, including a human, having cancer in an amount effective to treat the cancer in the animal. The unexpected and surprising ability of M-DNA and of MCC to induce cell cycle arrest in cancer cells, to inhibit proliferation of cancer cells, to induce apoptosis in cancer cells and to potentiate the antineoplastic effect of chemotherapeutic agents on cancer cells addresses a long felt unfulfilled need in the medical arts and provides an important benefit for animals, including humans.
Another object of the present invention is to provide a composition and method that induces cell cycle arrest in cancer cells.
Another object of the present invention is to provide a composition and method that inhibits proliferation of cancer cells.
Another object of the present invention to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent on cancer cells.
Another object of the present invention to provide a composition and method that potentiates the effect of a chemotherapeutic agent on cancer cells by synchronizing the cell cycle of the cancer cells.
Another object of the present invention is to provide a composition and method that potentiates the effect of a chemotherapeutic agent on proliferation of cancer cells.
Another object of the present invention is to provide a composition and method effective to treat cancer in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent in treating cancer in an animal, including a human.
Another object of the present invention is to provide a composition and method effective to eliminate cancer in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent in eliminating cancer in an animal, including a human.
Another object of the present invention is to provide a composition and method that induces cell cycle arrest in malignant melanoma cells.
Another object of the present invention is to provide a composition and method that inhibits proliferation of malignant melanoma cells.
Another object of the present invention to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent on malignant melanoma cells.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent on cell cycle arrest in malignant melanoma cells.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent on proliferation of malignant melanoma cells.
Another object of the present invention is to provide a composition and method that induces apopotosis in malignant melanoma cells.
Another object of the present invention is to provide a composition and method that activates caspases in malignant melanoma cells.
Another object of the present invention to provide a composition and method effective to treat malignant melanoma in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent in treating malignant melanoma in an animal, including a human.
Another object of the present invention to provide a composition and method effective to eliminate malignant melanoma in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the antineoplastic effect of a chemotherapeutic agent in eliminating malignant melanoma in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the effect of radiation in treating cancer in an animal, including a human.
Another object of the present invention is to provide a composition and method that potentiates the effect of radiation in eliminating cancer in an animal, including a human.
Another object of the present invention to provide a composition and method that potentiates the effect of radiotherapy on cancer cells by synchronizing the cell cycle of the cancer cells.
Another object of the present invention is to provide a composition that can be prepared in large amounts.
Another object of the present invention is to provide a composition that is relatively inexpensive to prepare.
Another object of the present invention is to provide a composition that has reproducible activity among preparations.
Another object of the present invention is to provide a composition that remains stable over time.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.